Background and Purpose The formation of reactive astrocytes is common following CNS injuries such as stroke. However, the signaling pathway(s) that control reactive astrocyte formation or functions are poorly defined. Here we assess the affects of Notch 1 signaling in peri-infarct reactive astrocytes after stroke. Methods We examined reactive astrocyte formation in the peri-infarct area 3 days following distal Middle Cerebral Artery Occlusion (dMCAO), with or without Gamma-secretase inhibitor (GSI) treatment. To directly study the effects of inhibiting a GS cleavage target in reactive astrocytes, we generated GFAP-CreER™∷Notch 1 conditional knock out (GN) mice. Results GSI treatment after stroke decreased the number of proliferative GFAP-positive reactive astrocytes and RC2-positive reactive astrocytes directly adjacent to the infarct core. The decrease in reactive astrocytes correlated with an increased number of CD45-positive cells that invaded into the peri-infarct area. To study the influence of reactive astrocytes on immune cell invasion, ex vivo immune cell invasion studies were performed. We found that a gamma-secretase mediated pathway in astrocytes affected Jurkat cell invasion. Following Tamoxifen (TM) treatment, GN mice had a significantly decreased number of proliferating reactive astrocytes and RC2-positive reactive astrocytes. TM treatment also led to an increased number of CD45-positive cells that invaded the peri-infarct area. Conclusions Our results demonstrate that proliferating and RC2-positive reactive astrocytes are regulated by Notch 1 signal transduction and control immune cell invasion after stroke.